JP6473001B2 - Large-scale deletion mutant of Aspergillus spp. - Google Patents

Description

  The present invention relates to Aspergillus spp. Mutants that retain a large deletion region near the telomere end of a chromosome homologous to chromosome 2 of Aspergillus oryzae.

  Aspergillus sojae and Aspergillus oryzae are widely used industrially for traditional food brewing such as soy sauce, sake, miso, enzyme production, and biomass utilization. Yes.

  In the manufacture of soy sauce, a traditional Japanese food, koji molds are grown on soybeans and wheat, which are raw materials, and koji is made. In koji and moromi, the protein, carbohydrates, and lipids of the raw soybeans and wheat are decomposed by the physical action of the koji mold and the enzymatic action of various hydrolases secreted by koji mold. Releases taste components such as amino acids, sugars, and glycerol. If the raw material decomposition in this process is improved, the raw material utilization rate and squeezability are increased, and the productivity can be improved.

  As described above, the breeding of koji molds that improve the raw material utilization rate and the squeezability is extremely important in industry, and breeding for this purpose has been vigorously carried out so far.

  An object of this invention is to obtain the koji mold which the viscosity of fermentation moromi falls and pressability improves.

  Surprisingly, the inventors have extensively deleted the telomere end of the chromosome homologous to the right arm of chromosome 2 of Aspergillus oryzae, thereby increasing hydrolase activity and, consequently, fermentation flavour. As a result, it was discovered that mutants of Aspergillus filamentous fungi having reduced viscosity and improved squeezability can be obtained, thereby completing the present invention.

That is, the present invention relates to the following aspects.
[Aspect 1]
A variant of a strain belonging to the genus Aspergillus, wherein at least part of the nucleotide sequence of the region from the telomere end of the chromosome homologous to the right arm of chromosome 2 of Aspergillus oryzae in the strain to 250 kb is deleted; and A mutant having increased hydrolase activity as compared with a strain not lacking the nucleotide sequence.
[Aspect 2]
In the region from the telomere end to 250 kb, the following regions i) to v):
i) a region from the telomere end to 50 kb;
ii) a region from the telomere end to 50-100 kb;
iii) a region from the telomere end to 100-150 kb;
iv) a region from 150 to 200 kb from the end of the telomere;
v) a region from 200 to 250 kb from the end of the telomere,
The variant according to aspect 1, wherein the nucleotide sequence of any one or more of the regions is deleted.
[Aspect 3]
The variant according to aspect 2, wherein the nucleotide sequence in the region of ii) and / or v) is deleted.
[Aspect 4]
The variant according to aspect 2 or 3, wherein the entire nucleotide sequence of the region from the telomere end to 250 kb is deleted.
[Aspect 5]
The mutant according to aspect 1, wherein the function of the tmpB (Trans membrane protein B) gene is reduced or deficient.
[Aspect 6]
The variant according to aspect 5, wherein a part or all of the tmpB gene is deleted.
[Aspect 7]
The mutant according to any one of aspects 1 to 6, wherein the strain belongs to Aspergillus sojae or Aspergillus oryzae.
[Aspect 8]
A culture of the mutant according to any one of aspects 1 to 7, or an extract from the culture.
[Aspect 9]
A method for producing a mutant according to any one of aspects 1 to 7, comprising a step of deleting at least a part of the base sequence of the region from the telomere end of the right arm of chromosome 2 to 250 kb in the strain belonging to the genus Aspergillus. Including.
[Aspect 10]
A method for producing the culture according to Aspect 8 or an extract from the culture, comprising the step of contacting the mutant according to any one of Aspects 1 to 7 with plant and / or animal raw materials. ,Method.
[Aspect 11]
The manufacturing method of aspect 10 with which the said raw material contains soybean and wheat.
[Aspect 12]
The production method according to aspect 10 or 11, wherein the culture or the extract from the culture is strawberry or moromi.
[Aspect 13]
A method for producing a hydrolyzate of an organic material, wherein the mutant according to any one of aspects 1 to 7 or the culture according to aspect 8 or an extract from the culture is brought into contact with the organic material. A manufacturing method including a process.
[Aspect 14]
The production method according to aspect 13, wherein the organic material contains a protein, a polysaccharide, or a lipid.

  According to the present invention, hydrolase activity of Aspergillus filamentous fungi can be remarkably improved by deleting the telomere end of the chromosome homologous to chromosome 2 of Aspergillus oryzae on a large scale. Such an improvement in hydrolase activity contributes to an increase in productivity and efficiency in the brewing and fermentation industries in which a raw material hydrolysis step is required. For example, when a mutant obtained as a result of such a large-scale deletion is used in a process for decomposing raw materials such as moromi, the amount of juice recovered by natural dripping from moromi increases, and the weight of straw is also effective. In particular, in brewing, if the moromi viscosity is high, the squeezability is reduced and the yield is reduced. By using this mutant, a brewing method in which the moromi viscosity is reduced and the squeezability is improved can be provided. However, since the mutant of the present invention or a culture thereof, or an extract from the culture can be widely used in a method for producing a hydrolyzate of an organic material, its use is not limited to the food field.

Method for producing a chromosome 250 kb deletion strain in Aspergillus soja NBRC4239 strain Confirmation of Homologous Recombination by Southern Hybridization of Chromosome 250kb Deletion in Aspergillus soja NBRC4239 Moromi viscosity and amount of residue in a short-term degradation test of a chromosome 250 kb deletion strain in Aspergillus soja NBRC4239 strain Residue amount and weight after pressing in soy sauce brewing of chromosome 250 kb deletion strain in Aspergillus soja NBRC4239 strain Moromi viscosity in a short-term degradation test of a chromosome 250 kb deletion strain in Aspergillus soja NBRC4241 strain Aspergillus soya NBRC4241 strain chromosomal 250 kb deletion strain weight after pressing in soy sauce brewing Method for producing Aspergillus soja NBRC4241 strain Δ50k_I strain Confirmation of homologous recombination by Southern hybridization of Aspergillus soja NBRC4241 strain Δ50k_I Method for producing Aspergillus soja NBRC4241 strain Δ50k_II Confirmation of homologous recombination by Southern hybridization of Aspergillus soja NBRC4241 strain Δ50k_II Method for producing Aspergillus soja NBRC4241 strain Δ50k_III strain Confirmation of homologous recombination by Southern hybridization of Aspergillus soja NBRC4241 strain Δ50k_III Method for producing Aspergillus soja NBRC4241 strain Δ50k_IV strain Confirmation of homologous recombination by Southern hybridization of Aspergillus soja NBRC4241 strain Δ50k_IV Method for producing Aspergillus soja NBRC4241 strain Δ100k strain Confirmation of homologous recombination by Southern hybridization of Aspergillus soja NBRC4241 strain Δ100k Miso Viscosity in a Short-term Decomposition Test with a Partially Deficient Strain in Aspergillus soja NBRC4241 Method for producing tmpB deletion strain in Aspergillus soja NBRC4241 strain Confirmation of homologous recombination by Southern hybridization and PCR of Aspergillus soja ΔtmpB strain Aspergillus soja moromi viscosities in a short-term decomposition test with ΔtmpB strain Method for producing chromosome 2 deletion mutant of Aspergillus oryzae RIB40 Confirmation of homologous recombination by Southern hybridization and PCR of Aspergillus oryzae RIB40 Residue amount of Aspergillus oryzae RIB40 by the short-term degradation test of chromosome 2 deletion strain 250kb Schematic diagram of Aspergillus soja Scaffold00063, various deletion regions and the tmpB gene Aspergillus soja tmpB gene and phylogenetic tree of orthologs in filamentous fungi

1. The present invention relates to a variant of a strain belonging to the genus Aspergillus, and a region from the telomere end of the chromosome homologous to the right arm of chromosome 2 of Aspergillus oryzae in the strain to 250 kb. And a mutant having an increased hydrolase activity as compared with a strain not having the base sequence deleted. The strains belonging to the genus Aspergillus include Aspergillus sojae, Aspergillus oryzae, Aspergillus niger, Aspergillus luchuensis, Aspergillus luchuensis, Aspergillus sperm Any of the strains of the strains belonging to Aspergillus soya and Aspergillus oryzae, and the group of genes existing from the telomere end of the chromosome homologous to the right arm of chromosome 2 of Aspergillus oryzae of these strains to 250 kb, In particular, a strain having the tmpB gene or an ortholog thereof is preferable.

  As a more specific strain, for Aspergillus soja, Aspergillus soya NBRC4239 strain (Deposit organization: National Institute of Technology and Evaluation, Biotechnology Headquarters, deposit number ID: NBRC4239) can be mentioned.

The mutant of the present invention can be prepared by extensively deleting the telomere end of a chromosome homologous to the right arm of chromosome 2 of Aspergillus oryzae, a strain belonging to the genus Aspergillus. Thereby, the hydrolase activity is increased compared to the parental strain which is not deleted. The deletion site may be at least a part of the base sequence in the region from the telomere end to 250 kb. For example, the following areas i) to v):
i) A region from the telomere end to 50 kb (corresponding to a base sequence of Δ50 k_I);
ii) A region from the telomere end to 50 to 100 kb (corresponding to a base sequence of Δ50k_II);
iii) a region from the telomere end to 100 to 150 kb (corresponding to a base sequence of Δ50k_III);
iv) A region from 150 to 200 kb from the telomere end (corresponding to a base sequence from the 5 ′ end of the Δ100 k region to 50 kb);
v) A region from the telomere end to 200 to 250 kb (corresponding to a base sequence of Δ50 k_IV),
It is assumed that at least a part of the base sequence of any one or more of these regions is deleted. The deletion of at least a part of the base sequence is a deletion that increases the hydrolase activity as compared with the parent strain even if the deletion is a single base.

  From the viewpoint of further increasing the hydrolase activity, it is preferable that the base sequence in the region of ii) and / or v) is deleted. More preferably, the entire nucleotide sequence of the region, ie, the entire 250 kb region counted from the telomere end of the chromosome homologous to the right arm of chromosome 2 of Aspergillus oryzae is deleted.

  The mutant of the present invention can be prepared by deleting an arbitrary region by genetic recombination using a vector in which both ends of an arbitrary region to be deleted are connected.

  The vector may include a marker gene to allow selection of transformed cells.

Examples of the marker gene include genes that complement the auxotrophy of the host, such as adeA , pyrG , argB , trpC , niaD , sC , and resistance genes for drugs such as pyrithiamine and aureobasidin.

Examples of the promoter used for forced expression of the target transcription control factor include alkaline protease promoter ( alp promoter), translation elongation factor promoter ( tef1 promoter), α-amylase promoter, and the like.

  The mutant of the present invention can be obtained by transforming a host with a recombinant vector. The host is not particularly limited as long as it is a filamentous fungus, and examples include Aspergillus sojae and Aspergillus oryzae.

  Transformation can be performed by a known method depending on the host. In the case of using a filamentous fungus, for example, Mol. Gen. Genet. 218: 99-104, 1989 can be used.

  Instead of transformation by the above deletion, a mutant having increased hydrolase activity can also be obtained by reducing or deleting the function of the tmpB (Trans membrane protein B) gene or its ortholog. The present inventors have shown that the tmpB gene exists as an ortholog in various Aspergillus spp. (FIG. 25), and that Aspergillus soja's tmpB is Soid-Raggi G, et al. Molecular Microbiology (2006) 59 It has been confirmed that it corresponds to a paralog of the Aspergillus nidulans tmpA gene described in (3). Here, tmpA is a transmembrane protein having NAD (P), FAD, and a heme-binding domain. Disruption of the tmpA gene suppresses conidia formation, and forced expression promotes conidia formation in liquid culture. It is known that Thus, the function of the tmpA gene is independent of hydrolase activity.

  Decreased or deficient in the function of the tmpB gene may be caused by destroying the gene, for example, deleting part or all of the gene, or inserting another gene such as a drug resistance gene in the middle of the gene. The gene can be modified so that it does not function normally.

2. Use of mutant The present invention further provides a culture obtained using the mutant. The culture containing the mutant of the present invention may be a raw material rich in protein, such as cereals, vegetables, etc. (eg, soybeans, wheat, etc.) containing vegetable proteins, and meats, fish, etc., containing animal proteins. It can be used in the hydrolysis step. As used herein, the term “culture” refers to a fermented product such as koji to which Aspergillus spp. Is added, and also a moromi obtained by using the Aspergillus spp.

  The timing of adding the mutant that can be produced by the method including the step of bringing the mutant into contact with the proteinaceous raw material is not particularly limited. As long as the effects of the present invention, for example, the improved hydrolase activity, are not impaired, the culture may be treated by adding bacteria other than Aspergillus in the course of preparation of the culture or before and after the preparation. Other steps can be appropriately determined by those skilled in the art depending on the desired application.

  The variants of the invention or their cultures are envisaged for use in various fields that need to be treated with improved hydrolase activity, particularly in the brewing and fermentation industries. For example, various fermented foods manufactured using Aspergillus spp., Such as soy sauce, miso, etc., when the mutants of the present invention are used in the production of the raw materials, the improvement of the raw material utilization rate and squeezability, and the final product Contributes to improving productivity. Not only in the food field, the mutant of the present invention or a culture thereof can be widely used in a method for producing a hydrolyzate of an organic material. As used herein, an “organic material” is a substance composed of oxygen, hydrogen, nitrogen atoms, etc., with carbon as the main element, regardless of natural or non-natural origin, It means a substance that can be decomposed by bacteria. For example, according to the mutant of the present invention or a culture thereof, food residues and biomass containing proteins, polysaccharides, lipids and the like that are decomposed by filamentous fungi are efficiently decomposed into their constituent units such as peptides and monosaccharides. be able to.

  EXAMPLES Hereinafter, although this invention is concretely demonstrated according to an Example, the technical scope of this invention is not restrict | limited at all by these description.

-Production of a Scaffold00063 telomere terminal 250 kb deletion strain in Aspergillus soja NBRC4239 strain.
As for Aspergillus aspergillus soya NBRC4239, the result of the draft genome analysis was published in 2011 (DNA Research; 18, 165-176, 2011), and the base sequence of the gonococcal scaffold is DDBJ / ENA. / Genebank accession numbers DF093557 to DF093585 are registered and disclosed.

  When the base sequence of Scaffold00063; 7550-259170 in the genome sequence of Aspergillus soja NBRC4239 strain was deleted, it was verified that it has an effect of improving the decomposition efficiency of plant raw materials.

Use strain Aspergillus sojae KP-del strain (ΔpyrG, Δku70) (Aspergillus sojae NBRC4239 share derivatives.). The Aspergillus soja KP-del strain ( ΔpyrG , Δku70 ) is a strain obtained through the following procedure.

The Aspergillus soja NBRC4239 strain was cultured on a medium containing perchloric acid to obtain a strain in which a natural mutation was introduced into the niaD gene and inactivated (Aspergillus soja ΔniaD strain). An Aspergillus soja ΔpyrG , niaD + strain was prepared by introducing a niaD gene into which no mutation was introduced into the pyrG locus of this ΔniaD strain.

The delta pyrG, the ku70 gene niaD + strains, then disrupted by pyrG gene, performs removal of pyrG gene, delta ku70, was obtained Aspergillus sojae P6-1-12 strain is delta pyrG strain.

Then, it removed delta ku70 by performing the niaD gene that has been introduced into the pyrG locus of the P6-1-12 strain, delta pyrG, were obtained ND-del strain is delta niaD strain. ND-del strain a mutant niaD gene present in, replacing the niaD gene mutation is not introduced, Aspergillus sojae KP-del strain (delta ku70 is a strain of state niaD gene was restored to wild type, delta pyrG Obtained).

Working medium PD medium (polypeptone 1%, dextrin 2%, dipotassium hydrogen phosphate 0.5%, sodium nitrate 0.1%, magnesium sulfate 0.05%, casamino acid 0.1%, pH 6.0), Czapek-Dox minimal medium (glucose 3.0%, potassium chloride 0.05%, sodium nitrate 0.2%, dipotassium hydrogen phosphate 0.1%, magnesium sulfate 0.05%, ferric sulfate 0.001 %, PH 6.0), Marz agar medium (malt extract 8%, agar 1.5%, pH 6.0)
When the auxotrophic strain was cultured, the uridine auxotrophic strain was added with uridine having a final concentration of 10 mM, and 1.2 M sorbitol was added to the medium for regeneration of the mutant.

Preparation of Scaffold00063 telomere-terminated 250 kb deletion cassette FIG. 1 shows an outline of homologous recombination by transformation and a 250 kb deletion vector of Scaffold00063 of Aspergillus sojae. Using the primers As250RF_pyr and As250RR, AsAs250LF and As250LR_pyr, AsPyrF and AsPyrR shown in Table 1, the arm region and the marker gene in homologous recombination were cloned by PCR from the genomic DNA of Aspergillus soja NBRC4239 strain.

  For PCR, KOD plus Neo (manufactured by TOYOBO) was used. The cloned DNA fragment was purified using Gel Extraction Kit (manufactured by QIAGEN). Fusion PCR was performed using the three DNA fragments.

Finally, Nested PCR was performed using As250F_nested and As250R_nested using the Fusion PCR product as a template to construct a 250 kb deletion vector.

The conidia suspension of the transformed host strain was inoculated into 100 mL of PD medium containing 10 mM uridine in a 500 mL Erlenmeyer flask and cultured at 30 ° C. for 16 hours with shaking, and then the cells were collected. The collected cells were washed with 0.7 M potassium chloride buffer solution, and 30 with 0.7 M potassium chloride buffer solution containing 1.5% Lysing Enzyme (manufactured by Sigma) and 0.3% Yatalase (manufactured by Ozeki). The protoplast was adjusted by gently shaking at 3 ° C. for 3 hours. The obtained protoplasts were washed with 1.2 M sorbitol buffer and then transformed by the protoplast PEG method.

Confirmation of homologous recombination About the obtained mutant, homologous recombination was confirmed by the Southern hybridization method. Probes used for Southern hybridization were prepared by PCR using As250RF_pyr and As250R_nested shown in Table 1, and using DIG DNA Labeling Mix (Roche) instead of ordinary dNTP.

  After the genomic DNA of the transformant was extracted, it was digested with PstI (manufactured by Takara). After separation by agarose gel electrophoresis, the mixture was transferred to Nylon Membranes, positively charged (Roche), and the membrane and probe were allowed to hybridize overnight, washed, blocked, CDP-Star, ready-to-use (Roche). The product was made to emit light using LAS3000 (manufactured by Fuji Film Co., Ltd.).

  As a result, it was confirmed that the target mutant was obtained (FIG. 2). This 250 kb deletion strain was designated as Aspergillus soja Δ250k strain.

-Soy sauce raw material short-term decomposition test using Δ250k strain A short-term decomposition test of soy sauce raw material using this fungus was conducted to examine whether this strain is effective in promoting the decomposition of raw materials in soy sauce brewing.

Effect on moromi viscosity in soy sauce raw material short-term decomposition test with Δ250k strain After mixing defatted soybeans, wheat and water in a ratio of 1: 1: 1.3, weigh 15 g, put in a 150 mL Erlenmeyer flask, cotton plug And sterilized by autoclave. This was inoculated with 2.7 × 10 ⁷ conidia of Δ250k strain or NBRC4239 Δku70 strain (negative control) and mixed well (incorporation). This was cultured at 30 ° C., and after 46 hours had passed from the filling, it was fermented. Each experiment was repeated three times.

  After loosening the brewed sample well with a sterilized shell, 15 mL of 28% saline solution sterilized by autoclaving was added and mixed well, and the mixture was tasted and covered with a rubber stopper. This was decomposed for 90 hours while shaking at 25 ° C. and 180 rpm.

  After completion of the decomposition reaction, the sample was transferred to a 25 mL polypropylene tube, and the moromi viscosity was measured with a rotational viscometer (Viscometer BLII manufactured by TOKIMEC). In addition, in order to measure the amount of residue of this decomposed product, a certain amount is separated into a mesh (sampler tech PP24) having an opening of about 1.0 mm, and after washing with water, the dry weight of the solid matter remaining on the mesh is measured. did.

  The results are shown in FIG.

  In the Δ250k strain, the moromi viscosity decreased by about 25% compared to the control. Further, the amount of residue of 1.0 mm or more was reduced by about 50% compared to the control. From this, it is considered that when the Δ250k strain is used, the decomposition of the soy sauce raw material is promoted, and the viscosity of moromi and the amount of residue can be reduced.

-Soy sauce small preparation test using Δ250k strain As shown in Example 2, it was confirmed that deletion of Scaffold00063 telomere terminal 250 kb in Aspergillus soya NBRC4239 promotes decomposition of soy sauce raw material. Therefore, a small-scale trial brewing was actually conducted to examine whether the squeezability would improve.

Effect on Moromi Viscosity in Soy Sauce Small Preparation Test with Δ250k Strain 25% saline was added to the koji made with koji lid and mixed well to make moromi. To this was added purely cultured soy sauce lactic acid bacteria Tetragenococcus halofilus and soy sauce yeast Digosaccharomyces luxi and fermented at 15 to 30 ° C. for 90 days. About the obtained soy sauce moromi, the residue weight after water washing was measured with the mesh (sample plastic tech PP24) of about 1.0 mm of opening, and the residual weight of mash after pressing with the Kamata type press was measured.

  In addition, although the small preparation test was done by 3 repetitions, three moromi | flavors were mixed in the compression test, and it was done by 1 series.

  The results are shown in FIG.

Similar to the short-term moromi degradation test, the weight of the koji after washing with water was reduced to about half in the Δ250k strain. Moreover, the weight of the paddy after pressing using the Kamata-type pressing machine was reduced by about 5%.
From the above, it was confirmed that when the Δ250k strain was used, decomposition of soy sauce raw material was promoted even in soy sauce brewing, and the amount of residue could be reduced.

・Verification of the effects of Scaffold00063 telomere-terminated 250 kb deletion strain on moromi viscosity and raw material degradation efficiency in Aspergillus soja NBRC4241 strain

  The data shown in Examples 1 to 3 are all data obtained with derivatives of Aspergillus soja NBRC4239 strain, but the strain of Aspergillus soja NBRC4241 strain, which is a strain different from NBRC4239 strain, was used as a host. Even in the case, it was verified whether the Scaffold00063 telomere terminal 250 kb deletion was effective.

  Aspergillus aspergillus soya KuU1 strain (Δku70, ΔpyrG strain; mutant strain having Aspergillus soya NBRC4241 strain as a parent strain) was used as a host, and a strain lacking 250 kb of telomere terminal side of Scaffold00063 was prepared. Since the gene sequence hardly changed in Aspergillus soya, the transformation cassette used in Example 1 was used as it was. The transformation was performed in the same procedure as that described in Example 1.

A short-term degradation test was conducted to evaluate whether the mutants were effective in degrading soy sauce raw materials. The short-term decomposition test was performed in the same procedure as the method described in Example 2 except for the decomposition conditions. The decomposition condition was that 15 mL of 28% saline was added to 15 g of sputum and mixed well, followed by decomposition at 20 ° C. for 42 hours. After the decomposition, the moromi viscosity was measured.
The results are shown in FIG.

  In the Δ250k strain using the NBRC4241 strain, the moromi viscosity was reduced by about 50% compared to the control. From this, even when the NBRC4241 strain is used, it is considered that the degradation of the soy sauce raw material can be promoted by deleting the telomere terminal 250 kb of Scaffold00063.

  Furthermore, it verified whether the same effect was acquired also in a soy sauce small preparation test. A soy sauce small preparation test was carried out in the same procedure as in Example 3, and the weight of the koji after pressing with the Kamata type press was measured using the obtained moromi.

  The results are shown in FIG.

  In the Δ250k strain using the NBRC4241 strain, the weight of the straw after pressing was about 4% lower than that of the control. From this, it was confirmed that even in the small preparation test, even when the NBRC4241 strain was used as in the case of the NBRC4239 strain, the degradation of the soy sauce raw material was promoted by deleting the telomere terminal 250 kb of Scaffold00063.

-Verification of the effect of a partially deleted strain of Scaffold00063 telomere terminal 250 kb region on soy sauce koji Aspergillus soya NBRC4241 strain on moromi viscosity From the results of Examples 1 to 4, Aspergillus soya has no Scaffold00063 telomere regardless of species It became clear that the degradation efficiency of the soy sauce raw material was promoted by deleting the terminal 250 kb.

  Therefore, in order to narrow down the region that affects the moromi viscosity in the 250 kb region, the effect on the moromi viscosity was examined using a strain that was divided into about 50 kb to 100 kb and deleted.

Preparation of Scaffold00063 telomere terminal 250 kb partial deletion cassette The outline of the partial deletion cassette is shown in FIG. 7, FIG. 9, FIG. 11, FIG. Cassettes were prepared as Δ50k_I to Δ50k_IV and Δ100k from the telomere terminal side, respectively.
FIG. 24 shows a positional relationship between Δ50k_I to Δ50k_IV and Δ100k. The primers used are shown in Tables 2-6.

The right arm of Δ50k_I is the right arm of Δ250k (As2
50RF_pyr and As250RR amplification products) were used, and Δ50k_IV and Δ100k Left Arms were the same as Δ250k Left Arms (As250LF and As250LR_pyr amplification products).

  The pyrG marker was also a fragment amplified using the same primer as Δ250k. Each of Left Arm (amplified product of primers LF and LR_pyr) and Right Arm (amplified product of primers RF_pyr and RR) was purified by gel extraction, and then subjected to Fusion PCR with three DNA fragments together with the pyrG marker gene fragment.

Finally, PCR was performed using the Fusion PCR product as a template and the primers for each nested PCR, and vectors for Δ50k_I to Δ50k_IV and Δ100k were constructed.

Confirmation of transformation and homologous recombination Transformation was carried out in the same manner as described in Example 1. The obtained mutant was confirmed for homologous recombination by Southern hybridization.

  The region of the probe used for Southern hybridization, the restriction enzyme used, and the expected fragment length are shown in FIG. 7, FIG. 9, FIG. 11, FIG.

The probe used in Δ50k_I is a fragment amplified using the primers of SEQ ID NO: 1 and SEQ ID NO: 2, and the probe used in Δ50k_II and Δ50k_III is a fragment of the full length PyrG marker amplified by the primers of SEQ ID NO: 5 and SEQ ID NO: 6. As for the probes used at Δ50k_IV and Δ100k, the fragments amplified using the primers of SEQ ID NO: 3 and SEQ ID NO: 4 were used in the same manner as in Example 1.

  The results of Southern hybridization are shown in FIG. 8, FIG. 10, FIG. 12, FIG. All mutants detected signals as expected, confirming that the target strain was obtained.

Measurement of Viscosity of Moromi Short-term Degradation Product Using Partially Deletion Strains A short- term degradation test was conducted to evaluate whether the mutants were effective for soy sauce raw material degradation. The short-term decomposition test was performed in the same procedure as described in Example 4 except for the decomposition conditions. After the decomposition, the moromi viscosity was measured.

  The results are shown in FIG.

  In the partially deleted strain in the 250 kb region, the viscosity in the short-term decomposition moromi decreased at Δ50k_II, Δ50k_IV, and Δ100k.

  The deletion region of Δ50k_IV is contained in Δ100k, and the moromi viscosity of both strains is reduced almost equally. Therefore, it is considered that the region of Δ50k_IV contains an important region in the reduction of moromi viscosity. It was.

  Further, each partial deletion alone did not reach the moromi viscosity of the strain in which the entire 250 kb was deleted. This suggests that the loss of moromi viscosity due to the deletion of 250 kb of taffomer end side of Scaffold00063 is largely related to the deletion of the factor that inhibits the reduction of moromi viscosity contained in the deletion region of Δ50k_II and Δ50k_IV. It was done.

-Verification of the effect of tmpB gene deletion on soy sauce koji Aspergillus soja NBRC4241 strain on moromi viscosity, and further narrowing down the region contained in the Δ50k_II region step by step, the result was the tmpB gene which is the Trans membrane protein (SEQ ID NO: 47) 48), the tmpB gene disruption strain was prepared and the effect was verified.

Preparation of tmpB gene deletion cassette The outline of the tmpB gene deletion cassette is shown in FIG. The primers used are shown in Table 7.

  Similarly, Left Arm, Right Arm and PyrG markers were amplified by PCR in the same manner as described above, and then each fragment was ligated by Fusion PCR. Finally, nested PCR was performed to construct a target tmpB deletion vector.

Confirmation of transformation and homologous recombination Transformation was carried out in the same manner as described in Example 1. The obtained mutant was confirmed for homologous recombination by Southern hybridization. For Southern hybridization, a PyrG marker region probe was used, and the genomic fragment was digested with BglII. The expected fragment is detected as a 7.4 kb fragment by the target recombinant product and a 3.0 kb fragment by the endogenous inactivated pyrG gene. As a result of Southern hybridization, expected bands for both ΔtmpB_1 and ΔtmpB_2 were detected. However, since ΔtmpB_1 also detected a signal in the vicinity of 4.5 kb, ΔtmpB_2 was used in the subsequent experiments (FIG. 19).

Viscosity measurement of moromi short-term degradation product by ΔtmpB strain A short- term degradation test was conducted to evaluate whether the mutants were effective in decomposing soy sauce raw materials. The short-term decomposition test was performed in the same procedure as described in Example 4 except for the decomposition conditions. After the decomposition, the moromi viscosity was measured. The results are shown in FIG.

  Disruption of the tmpB gene decreased the moromi viscosity by about 30%. From this, it was suggested that the deletion of the tmpB gene contributes to the improvement of the decomposition efficiency of the soy sauce raw material.

  However, since the moromi viscosity was reduced by about 60% in the strain lacking 250 kb, it was considered that the 250 kb region contained factors contributing to the degradation of the raw material in addition to the tmpB gene.

・Verification of effects of Scaffold00063 telomere terminal 250 kb and homologous region deletion strain on Aspergillus oryzae RIB40 strain on moromi viscosity and raw material degradation efficiency All the data shown in Examples 1 to 5 are from Aspergillus soya The same effect can be obtained by deleting a region homologous to Scaffold00063 telomere terminal 250 kb even when the Aspergillus oryzae RIB40 strain, which is the genome decoding strain of Aspergillus oryzae, is used as a host. We verified whether it was possible.

  First, homology analysis of the telomere terminal region of Scaffold00063 and the genome of Aspergillus oryzae RIB40 in Aspergillus soya NBRC4239 was performed.

  A search was performed using the BLAST program using the 450 kb telomere terminal region of Scaffold00063 in Aspergillus soja NBRC4239 as a query.

  As a result, there are several deletions in Aspergillus oryzae, which are about 100 kb shorter when viewed at 450 kb, but generally have homology with the end of SC003 on the right arm of chromosome 2. Was confirmed.

  Therefore, a cassette was prepared for deleting the SC003 region having homology with the 250 kb telomere terminal region of Scaffold00063 in the terminal Aspergillus soja NBRC4239 of SC003.

  The outline of the cassette is shown in FIG.

  Using the primers Ao250RF_pyr and Ao250RR, Ao250LF and Ao250LR_pyr, AoPyrF and AoPyrR shown in Table 7, the arm region and the marker gene in homologous recombination were cloned from the genomic DNA of Aspergillus oryzae RIB40 strain by PCR.

  For PCR, KOD plus Neo (manufactured by TOYOBO) was used. The cloned DNA fragment was purified using Gel Extraction Kit (manufactured by QIAGEN). Fusion PCR was performed using the three DNA fragments.

  Finally, Nested PCR was performed using Ao250F_nested and Ao250R_nested with the Fusion PCR product as a template to construct a vector for deletion of the SC003 telomere end 250k of Aspergillus oryzae RIB40.

Production of 250 kb deletion strain by homologous recombination Using the constructed vector, RKuN16prt1 (Δku70, ΔpyrG) strain, which is an auxotrophic host having improved transformation efficiency with Aspergillus oryzae RIB40 as a parent strain, was transformed. went. The control strain was produced by homologous recombination that restores the deleted pyrG gene.

  As a result of confirming homologous recombination in the obtained mutant by Southern hybridization, it was confirmed that the vector was incorporated at the target site as shown in FIG. However, since the same band as the parent strain was also detected, it was suggested that the obtained mutant was heterokaryon. Therefore, subculture was repeated in the Czapek-Dox minimal medium to purify the nuclei. Purification was confirmed by PCR using AoLF_t and AoLR_t primers.

  As a result, as shown in FIG. 22, since the PCR product derived from the parent strain disappeared in the mutant, acquisition of a 250 kb deletion strain of homokaryon was confirmed.

Short-term decomposition with a 250 kb deletion strain A soy sauce cake was prepared on a small scale using the above-mentioned deletion strain, and short-term decomposition was performed. After performing the decomposition at 30 ° C. for 42 hours, the amount of residue of the decomposition product was measured.

  As a result, as shown in FIG. 23, it was confirmed that the amount of residue of the degradation product in Aspergillus oryzae was reduced to about half compared to the control strain, as in Aspergillus soya.

Claims (9)

A variant of a strain belonging to the genus Aspergillus, wherein the tmpB (Trans membrane protein B) gene is disrupted and the hydrolase activity is increased compared to a strain in which the gene is not disrupted body. The mutant according to claim 1 , wherein the disruption is a deletion of the entire nucleotide sequence of the region from the telomere end of the chromosome homologous to the right arm of chromosome 2 of Aspergillus oryzae in the strain to 250 kb. The mutant according to claim 1 , wherein a part or all of the tmpB gene is deleted. The mutant according to any one of claims 1 to 3 , wherein the strain belongs to Aspergillus sojae or Aspergillus oryzae. Cultures variant according to any one of claims 1-4, or an extract from the culture. A method for producing a variant according to any one of claims 1-4, comprising the step of disrupting the tmpB gene in strain belonging to the genus Aspergillus, method. A method for producing the culture according to claim 5 or an extract from the culture, wherein the mutant according to any one of claims 1 to 4 is contacted with a plant and / or animal material. A method comprising the step of: The manufacturing method of Claim 7 with which the said raw material contains a soybean and wheat. The production method according to claim 7 or 8 , wherein the culture and the extract from the culture are koji or moromi.